家兔慢性细菌性鼻窦炎鼻黏膜创伤修复的研究

发布时间：2018-05-22 08:43

本文选题：家兔 + 动物模型　；参考：《复旦大学》2010年博士论文

【摘要】： 目的了解家兔鼻窦黏膜创伤修复的病理生理过程,并检测相关细胞因子的表达。方法以家兔作为研究对象,并用4mm电钻在上颌窦内侧壁造口制作创伤修复动物模型。分别于术后第2、4、6、8、10、14天各处死4只家兔,取上颌窦内侧壁记录造口的孔径,并用HE染色(Hematoxylin and Eosin Staining, HE Staining)和Masson三染色法观察再生鼻窦黏膜的形态学特征,酶联免疫吸附试验(Enzyme-Linked Immunosorbent Assay, ELISA)检测创伤修复过程中再生的鼻窦黏膜中IL-6、8、10以及Ⅰ型和Ⅲ型胶原的表达。结果家兔上颌窦内侧壁在创伤后第4天开始,其孔径明显缩小,并于第14天可完全愈合。再生的上颌窦黏膜中有较多的不成熟的胶原沉积,且以Ⅰ型胶原为主,而Ⅲ型胶原的比例有所下降。再生的部分上颌窦黏膜的上皮层缺乏纤毛分化,固有层中腺体减少,有时可见假性腺体结构。术后第14天再生的鼻窦黏膜中IL-6的表达较正常组明显升高(p0.0001)。术后第2、4、6、8、10天组间再生的鼻窦黏膜中IL-6表达的差异无明显统计学意义。术后各组再生的鼻窦黏膜中IL-8的表达明显升高(p=0.0011)。而术后各组再生的鼻窦黏膜中IL-10的表达与正常组比较明显降低(p=0.0003)。但术后第2、4、6、8、10、14天组间再生的鼻窦黏膜中IL-8和IL-10表达的差异没有明显统计学意义。与正常对照组相比,术后各组再生的鼻窦粘膜中Ⅰ型胶原的表达明显上升(p0.0001),而Ⅲ型胶原的表达明显降低(p0.0001)。相关分析表明,IL-6和IL-8的高表达与Ⅰ型胶原的过度沉积有关。结论家兔上颌窦黏膜创伤后再生的鼻窦黏膜中有较多的胶原沉积,并伴有促炎因子IL-6、8的高表达和抑炎因子IL-10的低表达。再生的鼻窦黏膜中的胶原蛋白以Ⅰ型胶原为主,且再生的鼻窦黏膜中过量的胶原沉积与IL-6、8的高表达以及IL-10的低表达有关。目的观察上调IL-10的表达对家兔慢性细菌性鼻窦炎动物模型上颌窦黏膜创伤修复的影响。方法以慢病毒为表达载体,上调IL-10的表达(LV-IL-10).实验分三组：生理盐水注射组、LV-IL-10治疗组和LV-GFP注射组。制作家兔慢性细菌性鼻窦炎动物模型,然后在其上颌窦内侧壁用4mm电钻进行造口制成创伤模型。在创伤前3天,各组上颌窦内侧壁黏膜下分别注射生理盐水、LV-IL-10和LV-GFP。于创伤后第3天和第10天取再生的上颌窦内侧壁黏膜用Real-time PCR法和酶联免疫吸附试验(Enzyme-Linked Immunosorbent Assay, ELISA)分别在mRNA水平和蛋白水平检测相关细胞因子的表达。HE和Masson三染色法观察创伤后第10天再生黏膜的形态特征。结果创伤后第3天和第10天再生的上颌窦黏膜中均可见慢病毒颗粒的表达,但后者明显低于前者。在创伤后第3天和第10天再生的上颌窦黏膜中,与生理盐水注射组和LV-GFP注射组比较,LV-IL-10治疗组的胶原沉积明显减少,炎症反应较轻,IL-6以及Ⅰ型和Ⅲ型胶原的mRNA表达水平明显下降,进一步ELISA法分析IL-6、IL-8以及Ⅰ型和Ⅲ型胶原的蛋白表达水平也明显下降,但三组间再生的上颌窦黏膜中TGF-p mRNA的表达无明显差别。结论在家兔慢性细菌性鼻窦炎创伤修复的动物模型中,上调IL-10的表达可以抑制再生黏膜中的炎症反应,减少胶原沉积,改善再生黏膜的组织重塑。
[Abstract]:Objective to understand the pathophysiological process of wound healing in rabbit nasal sinuses and detect the expression of cytokines.
Methods the rabbits were used as the research object, and the wound repair animal model was made with the 4mm drill in the medial wall of the maxillary sinus. 4 rabbits were killed on the 2,4,6,8,10,14 day after the operation. The aperture of the stoma was recorded in the medial wall of the maxillary sinus, and the regenerative nose was observed by HE staining (Hematoxylin and Eosin Staining, HE Staining) and Masson three staining. The morphological characteristics of sinus mucosa and Enzyme-Linked Immunosorbent Assay (ELISA) were used to detect the expression of IL-6,8,10 and type I and type III collagen in the sinus mucosa regenerated during the healing process.
Results the inner wall of the maxillary sinus began in the rabbit fourth days after the trauma. The pore size of the maxillary sinus was obviously narrowed and completely healed on the fourteenth day. There were more immature collagen deposits in the mucosa of the regenerated maxillary sinus and the type I collagen was dominant, and the proportion of type III collagen decreased. The epithelium of the maxillary sinus mucosa of the regenerated part was deficient in cilium differentiation and fixation. In the fourteenth day after operation, the expression of IL-6 in the sinus mucosa was significantly higher than that in the normal group (P0.0001). There was no significant difference in the expression of IL-6 in the sinus mucosa of the regenerated paranasal sinus after operation on day 2,4,6,8,10 (p=0). The expression of IL-8 in the paranasal sinus mucosa after the operation was significantly increased (p=0 .0011). The expression of IL-10 in the paranasal sinus mucosa after the operation was significantly lower than that in the normal group (p=0.0003). However, there was no significant difference in the expression of IL-8 and IL-10 in the sinus mucosa of the regenerated paranasal sinus after the operation on day 2,4,6,8,10,14. The expression of type III collagen increased significantly (P0.0001), while the expression of type III collagen decreased significantly (P0.0001). Correlation analysis showed that the high expression of IL-6 and IL-8 was related to excessive deposition of type I collagen.
Conclusion there is more collagen deposition in the sinus mucosa of the maxillary sinus mucosa after the maxillary sinus trauma in rabbits, with high expression of pro-inflammatory factor IL-6,8 and low expression of anti inflammatory factor IL-10. The collagen in the regenerated sinus mucosa is mainly collagen type I, and the excess collagen deposition in the regenerated sinus mucosa and the high expression of IL-6,8 and IL-10 It is related to low expression.
Objective to investigate the effect of up regulation of IL-10 on wound healing of maxillary sinus mucosa in rabbits with chronic bacterial sinusitis.
Methods the expression of IL-10 (LV-IL-10) was up-regulated with lentivirus as the expression vector. The experiment was divided into three groups: the saline injection group, the LV-IL-10 treatment group and the LV-GFP injection group. The rabbit model of chronic bacterial sinusitis was made, and then the wound model was made with the 4mm electric drill in the medial wall of the maxillary sinus. In the 3 day before the trauma, the maxillary sinus was in the maxillary sinus. The mucosa of the medial wall of the maxillary sinus was detected by Real-time PCR and enzyme linked immunosorbent assay (Enzyme-Linked Immunosorbent Assay, ELISA) on the medial wall of the maxillary sinus at third and tenth days after the trauma, respectively. The expression of.HE and Masson three were detected at mRNA level and protein level at the level of mRNA and protein, respectively, at third days and tenth days after the trauma. The morphological characteristics of regenerative mucosa on the tenth day after trauma were observed by color method.
Results the expression of lentivirus particles was found in the maxillary sinus mucosa of the maxillary sinus on third and tenth days after trauma, but the latter was significantly lower than the former. In the maxillary sinus mucosa of the third and tenth days after trauma, the collagen deposition in the LV-IL-10 treatment group was significantly reduced, the inflammatory reaction was lighter, IL-6 was lighter, IL-6 MRNA expression level of type I and type III collagen decreased significantly. Further ELISA analysis of IL-6, IL-8 and type I and type III collagen protein expression levels were also significantly decreased, but there was no significant difference in the expression of TGF-p mRNA in the three groups of regenerated maxillary sinus mucosa.
Conclusion in the animal model of rabbit chronic bacterial sinusitis wound repair, up regulation of IL-10 expression can inhibit the inflammatory reaction in the regenerated mucosa, reduce the deposition of collagen and improve the tissue remodeling of the regenerated mucosa.
【学位授予单位】：复旦大学
【学位级别】：博士
【学位授予年份】：2010
【分类号】：R765

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